Dataset Information

Overlap of formalin-fixed paraffin-embedded and fresh-frozen matched tissues for proteomics and phosphoproteomics

ABSTRACT: The proteome and phosphoproteome (PhosOme) of formalin-fixed paraffin-embedded (FFPE) tissues have potential for accelerating personalised medicine. Many liquid chromatography-mass spectrometry (LC-MS) studies have compared FFPE with matched fresh-frozen (FF) tissues to examine the effect of preservation techniques on the proteome however, few studies have included the PhosOme. Our aim was to quantitatively compare the proteome and PhosOme of FFPE and FF tissues using data-independent acquisition (DIA) LC-MS. To compare the two preservation techniques, four organs from three rats were cut in half to produce FFPE and FF matched tissue pairs. Across all organs, FFPE tissues gave less identifications than FF with an overlap of 85-92% for the proteome and 73-80% for the PhosOme. The proteome ratio of precursor summed intensity divided by the number of precursors was lower whilst the PhosOme ratio was slightly higher in FFPE tissues. Digestion efficiency was better in FFPE tissue thus the differences between the two preservation techniques must be a combination of FFPE block preanalytical factors (such as chemical modification of proteins) and lower MS peptide load. Hierarchical clustering demonstrated that biologically relevant information is retained in both preservation techniques with tissues clustering based on the organ. Strong agreement between FFPE and FF matched tissue pairs was observed with Pearson Correlation Coefficients of 0.89-0.97 and 0.79-0.87 for the proteome and PhosOme respectively. This data suggests that FFPE and FF preservation techniques are comparable for both proteomic and PhosOmic analysis.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Testis, Brain, Liver, Kidney

SUBMITTER: Erin Humphries

LAB HEAD: Phillip J Robinson

PROVIDER: PXD051873 | Pride | 2025-02-17

REPOSITORIES: pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20230106_EMH_F1-1_Prot_200ng_FFPE_Rat1_Brain_7600_ZS_1_03.htrms	Other
	20230106_EMH_F1-1_Prot_200ng_FFPE_Rat1_Brain_7600_ZS_1_03.timeseries.data	Other
	20230106_EMH_F1-1_Prot_200ng_FFPE_Rat1_Brain_7600_ZS_1_03.wiff	Wiff
	20230106_EMH_F1-1_Prot_200ng_FFPE_Rat1_Brain_7600_ZS_1_03.wiff.scan	Wiff
	20230106_EMH_F1-1_Prot_200ng_FFPE_Rat1_Brain_7600_ZS_1_03.wiff2	Wiff

Items per page:

1 - 5 of 797

Similar Datasets

Project description:This study was performed to evaluated RNA extraction and gene expression analysis of FFPE specimen stored for more than 20 years. Using long time stored FFPE material; large retrospective studies correlating molecular features with therapeutic response and clinical outcome, can be performed. Quantitative PCR (qPCR) was used to evaluate RNA extraction methods and to compare gene expression profiles of FFPE and fresh frozen (FF) tissue. Extracted RNA was subsequently subjected to microarray analysis and compared to qPCR data. The Ambion RecoverAll kit appears to be particularly suited for RNA extraction of long time stored FFPE tissues. Gene expression analysis using Affymetrix platform displayed a high degree of correlation for endogenous control genes comparing FF and FFPE tissues. We conclude that high quality gene expression signatures can be recognized using Affymetrix gene expression platform on FFPE tissue stored for more than 20 years. However, a general interpretation must be done with caution as different FFPE procedures have varying effects on RNA quality. Three RNA extraction methods from Roche (Basel, Switzerland), Ambion (Austin, TX, USA) and Qiagen (Hilden, Germany), designed for FFPE material was used. The methods were compared using qPCR. For the qPCR analysis, two different concentrations of input cDNA were used (20ng and 100ng) and 32 human endogenous control genes were examined. RNA from the Ambion FFPE kit was further analyzed by using microarray. Amplification of RNA prior to microarray analysis was performed using Nugen technologies (San Carlos, CA, USA). Nugen has developed amplification kits both for FFPE and FF materials: WT- ovation FFPE RNA amplification System V2 (Nugen FFPE), Ovation FF RNA Amplification System V2 (Nugen V2 FF) and Ovation FF Pico RNA Amplification System (Nugen PICO FF). The Nugen FFPE kit, designed for FFPE material was only used for FFPE tissue. The Nugen Pico FF kit, designed to target small amounts of FF RNA (>500 pg) and the Nugen V2 FF kit designed to target total FF RNA, were only used for FF tissue. Affymetrix standard amplification protocol (Affy FF) designed for FF RNA was also included as the standard method for amplification.

Project description:This experiment contains the RNA-Seq samples only. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5 % success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70-80 % of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.

Dataset Information

Overlap of formalin-fixed paraffin-embedded and fresh-frozen matched tissues for proteomics and phosphoproteomics

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets